Project 4: Molecular and cellular action of HLA class II molecules, the major genetic risk factors for VL The studies will build on observations made during the current TMRC, in collaboration with Phase 2 of the Wellcome Trust Case Control Consortium (WTCCC2), that HLA DR_DQ class 11 region contains the major genetic risk factor for visceral leishmaniasis in India (and Brazil). Four specific aims are proposed: 1. To determine transcriptional signatures in whole blood and/or splenic aspirates from active and past cases, and Quantiferon and/or antibody positive asymptomatic infected donors (Project 1), carrying risk versus protective HLA DRB1 allele groups. 2. To determine at mRNA and protein levels ex vivo, and following stimulation/infection of cells in vitro, whether cis-acting regulatory polymorphisms are functional correlates of risk and protective HLA DRB1 allele groups. 3. To purify, and identify using mass spectrometry, naturally processed leishmanial peptides from EBV transformed B cells prepared from HLA DRB1*13/*14 versus HLA DRB1*15 donors stimulated with whole crude parasite lysate prepared from a local Indian strain of L. donovani, compare with in silico predictions of epitopes and binding affinity, and develop a strategy to subvert the immune response by vaccination 4. To validate results of aim 3 by measuring antigen presenting cell function and immune response to naturally processed and predicted peptide epitopes in exposed individuals, including active and past cases, and asymptomatic infected donors. RELEVANCE (See instructions): Project 4. These studies will contribute to understanding of the molecular and cellular basis of HLA class genes as the major genetic risk factors for disease, and have implications for therapeutics and vaccine design and delivery.